This project is designed to enhance the PI's research capability and the skills and interests of her students in neuroscience by having them to participate in the PI?s ongoing research in the endogenous cannabinoid system at The University of Texas at Brownsville (an academically diverse 93% Hispanic community). Cannabinoids, the bioactive components in marijuana and hashish, affect the brain by acting at specific receptors, the brain cannabinoid receptors (CB1R). Natural ligands for CB1R are endogenous cannabinoids (eCBs). eCBs are increasingly recognized as critical signaling molecules in many areas of the nervous system because eCBs modulate many important neurobiological phenomena including neurotransmission, memory and learning, reward and motivation, and neuroprotection in diseases, which makes understanding the process of their synthesis a matter of great importance. However, the physiological stimuli that initiate the synthesis of eCBs are not well understood. The goal of this project is to investigate the cellular mechanisms of eCB production. Neuronal depolarization and the depolarization-induced increase in cytoplasmic calcium ([Ca2+]c) produce eCBs, and this production can be bio-assayed in real time by a phenomenon called DSI (depolarization-induced suppression of GABAergic inhibition). The PI?s preliminary findings suggest that Ca2+ release from the ryanodine receptor (RyR) determines the magnitude of DSI. From this evidence, it is hypothesized that RyR may be a key molecule that is necessary and sufficient for the production of eCBs in neurons. This hypothesis will be tested by accomplishing the following Specific Aims: 1) To isolate and characterize RyR-mediated [Ca2+]c signal and determine its role in the eCB production; 2) To identify the cellular localization of RyRs and their anatomical relationships with CB1R; and 3) To determine the roles of voltage-gated Ca2+ channels and store-operated Ca2+ entry in the RyR-dependent production of eCBs. Experiments are conducted in cultured hippocampal slices with the techniques of immunohistochemistry, siRNA and eGFP transfection, Ca2+ imaging, and the patch clamp recording. eCBs and their regulation by Ca2+ are topics of great interest to a wide range of biomedical scientists. Thus, the identification of Ca2+ signaling pathways and Ca2+ sensing proteins for the production of eCBs will provide a critical understanding for potent neuronal signaling systems, and open new avenues of investigations for the phenomena ranging from basic synaptic transmission to pathophysiological roles of eCBs. The project also offers a valuable opportunity for students to obtain laboratory research experiences, which undoubtedly raise their awareness in public health and diseases, and aid them in establishing their professional goals in biomedical sciences.